Method of producing l-glutamic acid



United States Patent 3,359,178 Patented Dec. 19, 1967 ice 3 359 178 METHOD OF PRODIjCID IG L-GLUTAMIC ACID Kat'sunobu Tanaka and Kazuo Kirnura, Machida-shi, and Ken Yamaguchi, Tokyo, Japan, assiguors to Kyowa tive and microbiological conversion of hydrocarbons as carbon source.

Briefly stated, the object of this invention is realized by the aforesaid expedient, the effective additament(s) medium containing such hydrocarbon(s). Still more particularly, the invention is concerned with improving the last-mentioned fermentative method of producing L-glutamic acid by the expedient of incorporating into the fermentation or nutrient medium an additament which functions to enhance the growth of the hydrocarbon-utilizing microorganisms with concomitant enhanced production of the desired L-glutamic acid.

A primary object of the present invention is the embodiment of a commercially feasible process for the microbiological or fermentative production of L-glutamic acid, using the readily available and correspondingly inexpensive source of carbon, namely, hydrocarbon or a mixture of hydrocarbons. Microorganisms are known which are capable of fermentatively converting hydrocarbon into amino acid. The quantity of amino acid produced is, however, so small-in reported cases of such conversionthat such processes can hardly be regarded as of significance in so far as the commercial production of glutamic acid is concerned. It is a desideratum in the art to be able to increase the yields of amino acid, and especially of L-glutamic acid, obtained by the fermenta- Hfaiyrko Kogyo Co., Ltd., Tokyo, Japan, a corporation being one or more of the following: o a an No D rawing. Filed Mar. 19, 1965, Ser. No. 441,339 Thiamine, Claims priority, application Japan, Mar. 23, 1964, p Aminobenzoic acid,

5574 Vitamin B 8 Claims. (Cl. 195Z8) iotln.

The effects of these vitamins for the growth of hydrocar- ABSTRACT OF THE DISCLOSURE hon-converting microorganisms with concomitant produc- L-glutamic acid is Produced from an aqueous nutrient tion of L-glutamic acid in a culture medium containing medium containing paraifinic hydrocarbon as carbon n-undecane as carbon source are shown in the following source by fermentation utilizing a microorganism capable table:

TABLE 1 Culture medium A Culture medium B Culture medium 0 Name of strains Growth Accumulation Growth Accumulation Growth Accumulation amounts, amounts of amounts, amounts of amounts, amounts of ing/m1. L-glutamic mg./ml. L-glutamic mg./ml. L-glutamic acid, mgJml. acid, mg./m1. acid, mgxlml Corynebacterium hydrocarboclastus N o. 2438 2. 4 0. 5 9. 8 5. 1 10. 1 5. 2 Arthrobacter simplex N o. 3151.. 2. O 0. 4 2. 1 0.5 10. 3 6.0 Micrococcus roseua N o. 401 2. 1 0.3 9. 5 5. 2 10.2 8. 1 Brevibacterz'um maria N o. 477 1. 9 0. 4 8. 3 5. 5 8. 6 10. 5

of converting hydrocarbon into L-glutamic acid, said Culture medium A: Wt. percent nutrient medium also containing not more than 207/ liter KH PO 0.1 of thiamine for enhancing the L-glutamic acid yield. Na HPO -12H O 0.1 MgSO -7H O 0.1 MnSO -4H O 0.002 This invention relates to the microbiological production 5 FeSO -7H O 0.02 of L-glutamic acid, and more particularly to a fermenta- (NH SO 2.0 tive method of producing L-glutamic acid, employing a n-Undecane 5.0 hydrocarbon or a mixture of hydrocarbons as carbon Phenol red 0.001 source and a microorganism which is capable of produc- CaCO 2.0 ing L-glutamic acid when cultured in an aqueous nutrient 40 Remainder water, pH 7.0.

Culture medium B: S'y/Iiter of thiamine is ture medium A. Culture medium .C: s'y/liter of thiamine, 0.1 m'y/liter of vitamine B 5'y/ml. of para-aminobenzoic acid and 0.3' /liter of biotin are added to Culture medium A.

(Culture conditions: 30 C., 220 r.p.m., 72 hours.)

The microorganism employed in carrying out the present invention is any one of those known to be able to fermentatively convert hydrocarbon into L-glutamic acid. Thus, use may be made more especially of microorganisms of the genera Corynebacterium and Arthrobacter, although as shown in Table 1 other hydrocarbon-converting microorganisms can also be employed.

As for the hydrocarbons used as assimilable carbon source in the process of the present invention, aliphatic hydrocarbons with 10 to 20 carbon atoms in the molecule can be used. However, best yields of L-glutamic acid are obtained with normal paraffins with 10 to 18 carbon atoms. Thus, useful hydrocarbons for the purposes of the present invention comprise decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane,

added to Culheptadecane, octadecane, nona'decane and eicosane, as the normal compounds or in the iso-forms, individually or in admixtures such as are frequently obtained in practice. However, the normal parafiins: n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadccane and n-octadecane, severally or in admixture, eg in the form of commercial mixtures, give superior results. Hydrocarbons with 11 and 12 carbon atoms are preferred.

As to the previously-enumerated additaments, the amounts thereof are not specially limited. However, since in some circumstances, overgrowth thereof at the expense of the production of objective L-glutamic acid may occur, the most efiicient ranges of said additaments are to l0'y/liter of thiamine;

0 to 50'y/ milliliter of p-aminobenzoic acid; 0 to 1 mxy/ liter of vitamin B 0 to liter of biotin.

The following table illustrates this in connection with thiamine, by way of example:

TABLE 2 Amounts of Accumulation thiamine in Growth amounts, amounts of the culture mg. m L-glutamic acid, medium, 'y/lilIB! mgJml.

Strain employed: Corynebacterium hydrocarboclastus No. 243-8.

Composition of culture medium:

KH PO weight percent 0.1 Na HPO l2H O do 0.1 MgSO -7H O do.. 0.1 MIISO4'4H2O ..dO- FeSO -7H O do 0.002 (NH4)2SO4 dO n-Undecane do 5 Phenol red do 0.001 CaCO do 2.0 Thiamine -y/liter 0-50 Remainder water, pH 7.0.

Examplel 20 milliliters of culture medium consisting of of KHZPOI,

0.1% Of Na2HPO4'12H20, 0.1% of MgSO -7H O, 0.002% of FeSO '7H O, 0.002% of MnSO 4-H O, 5 liter of thiamine,

20% of (NI-1.9 80

5% of n-unclecane, and 0.001% of phenol red, Remainder water, at pH 7.0,

were prepared in a conical flask with 250 ml. capacity, Corynebacterium hydrocarboclastus No. 2438 (ATCC No. 15592) was inoculated and incubated aerobically at 30 C. for 72 hours with shaking at 220 r.p.m. The accumulation amounts of L-glutamic acid in the culture liquor was 15.5 mg./ml. at the completion of cultivation. Thereafter L-glutamic acid was absorbed through cation exchange resin (Amberlite IR/20) and was eluted with dilute aqueous ammonia. About 220 milligrams of L-glutamic acid hydrochloride was obtained from the efiluent according to the conventional method.

On the other hand the accumulation amounts of L-glutamic acid in the culture medium was only 0.2 mg./ml. without the addition of thiamine by the same method as above.

ExampleZ To the same culture medium as in Example 1 in which 5% of kerosene was used as carbon source, Corynebacterium hydrocarboclastus No. 2438 was inoculated and aerobically incubated at 30 C. with shaking at 220 r.p.m. for 72 hours. About 35 mg. of L-glutamic acid hydrochloride was obtained by recovering 3.0 mg./ml. of L-glutamic acid which was accumulated at the completion of cultivation according to the same method as in Example 1.

Less than 0.1 mg./ml. of L-glutamic acid was produced by the similar culture method without thiamine.

Example 3 Arthrobacter simplex No. 3151 (ATCC No. 15799) was inoculated into 20 ml. of a culture medium consisting of 0.1% of KH PO of Na HPO 12H20, 0.1% of MgSO -7H O, 0.002% of FeSO -7H O, 0.002% of MnSO -4H O, 5'y/liter of thiamine,

0.1 mxy/l. of vitamin B 20% of (NH SO 5% of n-undecane, and 0.001% of phenol red Remainder water, at pH 7.0,

prepared in a conical flask with 250 ml. capacity and was incubated at 30 C. with shaking at 220 r.p.m. for 72 hours. 5.4 mg./ml. of L-glutamic acid was accumulated at the end of the cultivation. About 67 mg. of L-glutamic acid hydrochloride was obtained according to the method of Example 1.

When the culture medium without thiamine, vitamin B or both of them is employed the accumulation amounts of L-glutamic acid are less than 0.1 mg./ml. respectively.

What is claimed is:

1. In a method for producing L-glutamic acid from hydrocarbon by fermentation of an aqueous nutrient medium containing parafiinic hydrocarbon as carbon source and a microorganism capable of converting hydrocarbon into L-glutamic acid, the improvement according to which the nutrient medium contains an amount of thiamine sutficient to enhance the yield of L-glutamic acid, such amount being less than 207 of thiamine/liter.

2. The improvement according to claim 1, wherein the microorganism belongs to the genus Corynebacterium.

3. The improvement according to claim 1, wherein the microorganism belongs to the genus Arthrobacter.

4. The improvement according to claim 1, wherein the carbon source is constituted by kerosene.

5. The improvement according to claim 1, wherein the carbon source is constituted by n-undecane.

6. The improvement according to claim 1, wherein the microorganism is Corynebacterium hydrocarboclaslus (ATCC No. 15592) and the culture medium contains 5 S'y/Iiter of thiamine and 5% of n-undecane as sole carbon source.

7. The improvement according to claim 1, wherein the microorganism is Coryneb'acterium hydrocarboclastus (ATCC No. 15592) and the culture medium contains 5'y/liter of thiamine and 5% of kerosene as sole carbon source.

8. The improvement according to claim 1, wherein the microorganism is Arthrobacter simplex (ATCC No. 15799) and the culture medium contains S'y/Iiter of thiamine and 0.1 mr /liter of vitamin B and, as sole carbon source, 5% of n-undecane.

6 References Cited Shiio et 211.: Journal of General Applied Microbiology, vol. 9, N0. 1, 1963, pages 23 to 30.

LIONEL M. SHAPIRO, Primary Examiner.

A. LOUIS MONACELL, Examiner.

L. M. SHAPIRO, Assistant Examiner. 

1. IN A METHOD FOR PRODUCING L-GLUTAMIC ACID FROM HYDROCARBON BY FERMENTATION OF AN AQUEOUS NUTRIENT MEDIUM CONTAINING PARAFFINIC HYDROCARBON AS CARBON SOURCE AND A MICROORGANISM CAPABLE OF CONVERTING HYDROCARBON INTO L-GLUTAMIC ACID, THE IMPROVEMENT ACCORDING TO WHICH THE NUTRIENT MEDIUM CONTAINS AN AMOUNT OF THIAMINE SUFFICIENT TO ENHANCE THE YIELD OF L-GLUTAMIC ACID, SUCH AMOUNT BEING LESS THAN 20$ OF THIAMINE/LITER. 